Escherichia coli secreted protein b

ABSTRACT

Several EHEC proteins which are secreted into the culture supernatant have been discovered. These proteins are not produced by non-pathogenic  E. coli , and produce a strong serum antibody response in patients with HUS and bloody diarrhea.

FIELD OF THE INVENTION

[0001] The present invention relates to novel proteins which aresecreted from enterohemorrhagic Escherichia coli (EHEC), a pathogenresponsible for bloody diarrhea and hemolytic uremic syndrome (HUS) inhumans. The invention relates to serodiagnostic techniques of theseconditions using these proteins.

BACKGROUND OF THE INVENTION

[0002] The most common cause of bloody diarrhea and hemolytic uremicsyndrome (HUS) in North America is infection by enterohemorrhagic E.coli (EHEC) (1). Alternative names for EHEC are Shiga toxin-producing E.coli (STEC), Shiga-like toxin-producing E. coli (SLTEC),Verocytotoxin-producing E. coli (VTEC), or Verotoxin-producing E. coli.In the United States, this food-borne E. coli is the most commoninfectious cause of bloody diarrhea in individuals of all ages. HUS isthe most common cause of kidney failure in children in the U.S. andCanada.

[0003] This organism was the cause of the infamous “Jack-in-the-Box”food-poisoning outbreak in Seattle in 1993 which infected over 500people and resulted in 4 deaths and many cases of long-term kidneydamage. In 1996, this organism caused an enormous outbreak involvingmore than 8,000 people in Japan, resulting in 7 deaths. In late 1996,EHEC again caused an outbreak of food-poisoning in Scotland whichaffected 250 people and killed 18 people.

[0004] The most important virulence factor of E. coli associated withHUS is a potent cytotoxin known as Shiga toxin, Shiga-like toxin,verocytotoxin, or verotoxin, also called Stx. After production by E.coli colonizing the large intestine, Shiga toxin is absorbed into thecirculation and eventually affects the kidney. The evidence linking Stxto HUS is both epidemiological and experimental. The firstepidemiological association between Stx-producing E. coli and HUS wasmade by Karmali et al. (2) in 1983, and numerous studies since then havesupported this association. Subsequent in vitro studies have shown thatpurified Stx has profound effects on renal endothelial cells resultingin cell death (3).

[0005] However, besides the clear-cut involvement of Stx inpathogenesis, little is known about other bacterial virulence factorsinvolved in this disease. The lipopolysaccharide (LPS) of EHEC has beenreported to enhance the effect of the Stx on human vascular endothelialcells (4), although the exact mechanism is not known. The othervirulence factor of this organism that has been implicated in animalmodels is the 94 kilodalton outer membrane protein (OMP) known asintimin, which was discovered in the inventor's laboratory (5, 6).Intimin is involved in the colonization of the intestinal tract, whichis apparently necessary for disease, but there is no evidence thatintimin is directly involved in the renal disease. Since oral ingestionof preformed toxin is apparently not sufficient for causing HUS, otherbacterial virulence factors clearly must be involved in the pathogenesisof this disease.

[0006] Two Distinct Sites of Disease, Intestinal and Renal

[0007] Disease due to EHEC starts by ingestion of meat, water, or otheritems that are contaminated with this organism. The organism thencolonizes the large bowel where it can produce non-bloody diarrhea orbloody diarrhea (hemorrhagic colitis). In the colon, EHEC producesmucosal edema, erythema, ulceration and hemorrhage. A characteristichistopathology known as attaching and effacing (AE) results which ischaracterized by effacement of intestinal microvilli, intimate adherenceof bacteria to enterocytes, and accumulation of polymerized actin andother cytoskeletal components in the epithelial cell directly beneaththe adherent bacteria. The AE lesion has been repeatedly demonstrated inanimals infected with EHEC and in cultured human epithelial cells. AE isassumed to occur in the colon early during the course of humaninfection, although direct evidence is lacking, probably becausepatients with EHEC infections undergo colonoscopy relatively late in theinfection when the colonic surface has been denuded of epithelial cells.The AE histopathology is similar to that seen with enteropathogenic E.coli (EPEC), which do not produce Stx. We have shown that formation ofthe AE lesion by both EPEC and EHEC is mediated by the gene products ofa 35 kilobase region of chromosomal DNA present in these strains butabsent from normal flora E. coli (5, 7).

[0008] The pathogenic mechanisms by which EHEC produces non-bloodydiarrhea and bloody diarrhea (hemorrhagic colitis) are largely unknown.Formation of the AE lesion in the absence of Stx is believed to besufficient to cause non-bloody diarrhea in EPEC infections. Pure Stx canact as an enterotoxin, and in studies using rabbit jejunal tissuemounted in Ussing chambers, Stx selectively kills the absorptive tipcells while not affecting the secretory crypt cells, thereby changingthe net balance of secretory/absorptive processes towards secretion (8).The bloody diarrhea is presumably due at least in part to the powerfulcytotoxic effects of Stx, although the contribution of other bacterialfactors and the host inflammatory response is not known. In at least twoanimal systems, gnotobiotic piglets and rabbits, Stx was not requiredfor EHEC strains to alter secretory activity or cause severehistological changes (9, 10). O'Loughlin and colleagues (9, 11) haveshown that disruption of colonic epithelium and changes in electrolytetransport during EHEC infection in rabbits are mediated by the hostinflammatory response and that bacterial products other than Stx andfactors encoded on the 60 MDa plasmid (see below) are necessary for theintestinal manifestations of EHEC disease.

[0009] The classic HUS triad includes microangiopathic hemolytic anemia,thrombocytopenia, and renal failure and may be accompanied by centralnervous system manifestations in 30-50% of patients (12). Althoughhemolytic uremic syndrome (HUS) occurs in only 2-7% of all EHECinfections (while bloody diarrhea occurs in 90% of all infections(1,13)), HUS is associated with the greatest mortality due to thisorganism. The Shiga toxin produced in the bowel reaches the circulationand produces vascular endothelial damage that results in occlusion ofthe renal glomerular microvasculature by fibrin and platelets. Inductionof inflammatory cytokines has also been suggested to contribute to thedisease process (14-16). Although the preeminence of Stx in the diseaseprocess is accepted, the role of other bacterial factors in producinghost damage or facilitating the delivery of Stx is unknown.

[0010] Virulence factors of E. coli O157:H7

[0011] The majority of work on pathogenic factors of EHEC has focused onthe Shiga toxins, which are encoded on bacteriophage inserted into thechromosome. Additional potential virulence factors are encoded in thechromosome and on a 60 MDa plasmid found in most strains of EHEC

[0012] Toxins Stx occurs in two major forms, Stx1 and Stx2, which share55 and 57% sequence identity in the A and B subunits, respectively (17).While Stx1 is highly conserved, sequence variation exists within Stx2.The toxins consist of a single A subunit of ca. 32 kDa and 5 identical Bsubunits of ca. 7.7 kDa (18) The B subunit serves to bind the toxin to aspecific glycolipid receptor, globotriaosylceramide or Gb₃, while the Asubunit is internalized and cleaves N-glycoside bonds on the 28S rRNA ofthe 60S ribosome. The resulting disruption of protein synthesis leads todeath of renal endothelial cells, intestinal epithelial cells, vero orHela cells, or any cell which possesses the Gb₃ receptor.

[0013] The 60 MDa plasmid commonly found in EHEC strains contains genesencoding an -hemolysin (19). Although this hemolysin is widelydistributed among Stx-producing strains of E. coli, there are no dataindicating that it is expressed in vivo or involved in pathogenesis ofdisease. Two other distinctly different phage-encoded hemolysins, termedenterohemolysins, are produced by many Stx-producing E. coli (20, 21)but again, there are no data to suggest in vivo expression or any rolein pathogenesis.

[0014] Intestinal Adherence Factors

[0015] The only potential EHEC adherence factor which has beendemonstrated to play a role in intestinal colonization in vivo in ananimal model is the outer membrane protein intimin, encoded by the eaeAgene, also known as eae. We prepared an isogenic derivative of an EHECstrain specifically mutated in eaeA. In both conventional andgnotobiotic piglets (5, 22), a functional intimin protein was necessaryfor intimate adherence to intestinal epithelial cells, formation of theAE lesion, and induction of diarrhea. The importance of intimin in theseprocesses was independently confirmed by other investigators using adifferent EHEC eaeA mutant in gnotobiotic piglets (23).

[0016] Other candidate adhesins have been reported but none have beenwell characterized or specifically demonstrated to play a role inadherence in vivo. Sherman et al. (24) reported that a 94 kDa OMPdistinct from intimin (25) mediated adherence to Hep-2 epithelial cells,but no further characterization of this factor has been reported.Strains of EHEC produce fimbriae which might aid intestinal adherence(26-29), but no purified fimbriae or cloned fimbriae genes have beenreported. An initial report (27) suggested that the 60 MDa plasmid wasrequired for expression of fimbriae and adhesion to epithelial cells,but subsequent studies have reported that loss of the plasmid eitherenhanced adhesion (28), decreased adhesion (24), or had no effect onadhesion (29). The potential role of lipopolysaccharide (LPS) inadhesion was examined, and loss of LPS actually increased adherence tocultured epithelial cells (31). The existence of intestinal adherencefactors distinct from intimin is suggested by the isolation ofStx-producing E. coli strains of serotypes other than EHEC that lack theeaeA gene but are still associated with bloody diarrhea or HUS inhumans.

[0017] Other Potential Factors

[0018] LPS may either enhance or inhibit the toxicity of Stx in animalmodels (32,33) and enhances the cytotoxicity of Stx on human vascularendothelial cells in vitro (4). However, this effect is not specific forEHEC LPS since LPS from several species of the Enterobacteriaceae havesimilar effects (4). There is one report (34) that EHEC can invadecultured intestinal cell lines, but a later report (35) disputed thesefindings, showing that EHEC strains were no more invasive than normalflora E. coli. Furthermore, there is no in vivo evidence that invasionoccurs in humans or in animals.

[0019] Non-0157:H7 STEC

[0020] Enterohemorrhagic E. coli O157:H7 is the most important type ofE. coli that can cause HUS and bloody diarrhea. The O157:H7 nomenclaturerefers to particular bacterial surface antigens that define a serotype.The “O” designation refers to the surface lipopolysaccharide and the “H”antigen refers to the bacterial flagellar protein. Those E. coli thathave the O157 and H7 antigens have the full array of virulence factors(Stx and intimin) and are always considered to be pathogens (36).However, there are similar E. coli that do not have the O157:H7 serotypebut can cause similar disease. These E. coli are of serotypes such asO26:H11 and are called non-O157:H7 STEC (or non-O157:H7 EHEC). The majorpotential virulence factor that these organisms share is the Stx toxinbut not all non-O157:H7 STEC are pathogenic (36). Mere expression of Stxalone does not define a pathogen. In fact, up to 63% of meat samples inU.S. supermarkets contain non-O157:H7 STEC (36). In contrast, E. coliO157:H7 is rarely found in foods. When non-O157:H7 STEC are isolatedfrom patients, it is considered a pathogen but when they are isolatedfrom food or animals, their significance is unknown (36). The inventiondescribed herein aids the discrimination of non-O157:H7 STEC that are ofpublic health significance from those that are of little or no publichealth significance.

[0021] The reservoir of E. coli O157:H7 and non-O157:H7 STEC is believedto be in animals, particularly cattle (1). However, O157:H7 is rarelyisolated from cattle, usually from less than 1% of cattle in mostsurveys (1). In contrast, non-O157:H7 STEC can be isolated from up to40% of cattle (1). One important difference between non-O157:H7 STECthat are isolated from cattle and non-O157:H7 STEC isolated from humansis possession of the eae gene. The majority of these strains that areisolated from human disease possess the eae gene (37) whereas only aminority of these strains that are isolated from cattle possess eae andare presumably pathogenic for humans. Possession of the eae genecorrelates with possession of a large block of virulence genes calledthe LEE (7) that also encode secreted proteins that are the basis of thepresent invention.

[0022] Serologic Response

[0023] Determining the complete immune response to an infecting agenthas many applications for understanding and controlling an infectiousdisease. First, such information can be used in serdiagnostics andseroepidemiology studies to detect evidence of an infection where theactual infectious agent cannot be detected either because of the lack ofan appropriate specimen or lack of sufficiently sensitive detectionmethods. Second, knowledge of the components of an infectious agentwhich engender an immune response can be directly applied to developinga vaccine or immunotherapeutic agent (e.g., passive immunoglobulin).Third, detection of an immune response to a particular component of theinfectious agent is direct evidence that this component is actuallyexpressed in vivo during the course of infection, supporting thepossibility that it plays a role in the pathogenesis of disease. Theconclusion that an immune response indicates in vivo expression of theantigen is not always valid if, for example, the antigen is a toxin thatis preformed at the time of ingestion or the infectious agent directlyenters the blood stream without replication. However, for an agent thatis ingested and replicates in the intestine, an immune response to acomponent strongly implies in vivo expression. For these reasons, afuller determination of the immune response to E. coli O157:H7 isimportant for a better understanding of this disease.

[0024] Previous studies of the human immune response to E. coli O157:H7have focused on the serological response since there is no evidence thatcell-mediated immunity plays a role in this disease. The most widelystudied antigen is the LPS. Chart et al. (38) showed that in one studyof 60 patients with HUS, Stx or a Stx-producing E. coli could bedetected in only 23% of fecal specimens whereas an IgM response to theO157 LPS was detected in 73% of these patients. In an epidemicsituation, an immunoassay based on IgG responses to LPS was over 90%sensitive and specific for patients with recent culture-confirmedinfection (39?). Antibodies to the other antigen of the O157:H7serotype, the H7 flagella, were not detected in any of the HUS patientsstudied by Chart et al. (40). In this same study, some patients showedresponses to outer membrane proteins (OMPs) but this response was foundto be due to contaminating LPS co-migrating with the OMPs.

[0025] Stx represents an obvious choice for an important antigenproduced in vivo. However, numerous investigations of the serologicalresponse to Stx have yielded disappointing results. A curious phenomenonis that sera from most individuals without any history of infection withStx-producing E. coli contain a substance that is capable ofneutralizing Stx2. When ELISA rather than neutralization tests areconducted to detect antibodies against either Stx1 or Stx2, only aminority of HUS patients showed a response to these toxins (39, 41-43).Similar results are seen in patients infected with Stx-producingShigella dysenteriae type (44) and may be related to the fact that Stxis cytotoxic to human B lymphocytes (45). Whatever the reason, theseresults have led experts to conclude that antitoxin response is not auseful tool in the serodiagnosis of Stx-producing E. Coli (39, 41, 42,46)

[0026] Serological responses to E. coli O157:H7 and non-O157:H7 STEC incattle have not been as extensively studied as in humans. Some cattleexperimentally infected with E. coli O157 developed serum antibodiesagainst Stx and O157 LPS but others did not (55). Detection ofantibodies against Stx in cattle is not useful in predicting thepotential for human disease since the prevalance of non-O157:H7 STEC ofdoubtful pathogenic significance is so high in cattle, as describedabove.

[0027] In summary, several years of research into the pathogenesis andimmune response of E. coli O157:H7 infections have not yielded firmknowledge of the protective immune response or an ideal serodiagnostictool. The O157 LPS is useful for serodiagnosis, but it is difficult toprepare and the response may be nonspecific since O157 LPS sharesepitopes with E. coli O44 LPS (43) and the LPS of certain serogroups ofSalmonella spp., Yersinia enterocolitica, Brucella abortus, and Vibriocholerae non-O1 strains (reviewed in (47). It is also useless fordetecting infection due to Stx-producing E. coli strains of non-O157serogroups. On the other hand, Stx is clearly produced in vivo and isessential for disease, but is not useful for serodiagnosis.

SUMMARY

[0028] Although extensive research has been conducted on the Shigatoxins expressed by this organism, there is very little known aboutother potential virulence factors of this pathogen. There is clearly aneed in the art to have definitive diagnostic techniques available. Wehave discovered novel proteins that are secreted by Stx-producingstrains of E. coli serotypes O157:H7 and O26:H11. These proteins areexpressed during the course of infection and are highly immunogenic inhumans, and is therefore useful as serodiagnostic tool. Furthermore,these proteins are also immunogenic in cattle. The invention teaches amethod for determining cattle which are infected with EHEC. Cattle whichtest positive can be omitted from food production.

SUMMARY OF THE INVENTION

[0029] It is the object of the invention to provide proteins which aresecreted from EHEC, are called Esp, and trigger a strong immune responsein a subject.

[0030] It is another object of the invention to provide a 37 kilodaltonprotein (EspB) secreted from E. coli 0157:H7.

[0031] It is another object of the invention to provide a 24 kilodaltonprotein (EspA) secreted from E. coli 0157:H7.

[0032] It is another object of the invention to provide genes whichencode the secreted Esp proteins from E. coli 0157:H7.

[0033] It is a further object of the invention to provide aserodiagnostic test such as an ELISA or a Western blot for determiningthe presence of the secreted Esp proteins in a subject, wherein thesubject can be a human or a cow.

[0034] It is a still a further object of the invention to provide a testkit for both humans and cows which with the use of a substrate coatedwith the secrected Esp proteins, will diagnose the presence ofantibodies to the E. coli secreted Esp proteins in said human or cowalmost instantaneously.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1: Coomasie blue-stained SDS/polyacylamide gel (1A) andidentical immunoblot probed with rabbit antiserum against EPEC secretedproteins (B) of culture supernatants from EHEC strains 84-289 [O157:H7](lane 1), 85-170 [O157:H7] (lane 2), 86-24 [O157:H7] (lane 3), EDL933[O157:H7] (lane 4), NF4 [O157:H7] (lane5), 83-574 [026:H11] (lane 6),6549 [026:H11] (lane 7), EPEC E2348/69 (lane 8), and E. coli HS-4 (lane9).

[0036]FIG. 2: Immunoblot or culture supernatants from EHEC strains84-289 [O157:H7] (lane 1), 85-170 [O157:H7] (lane 2), 86-24 [O157:H7](lane 3), EDL933 [O157:H7] (lane 4), NF4 [O157:H7] (lane 5), 83-574[026:H11] (lane 6), 6549 [026:H11] (lane 7), EPEC E2348/69 (lane 8, andE. coli HS-4) probed with human serum from a patient who developed HUS.

[0037]FIG. 3: Immunoblot of secreted EHEC proteins probed with seracollected from a patient (HS363) infected with O157:H7 without HUS(panel A) and a patient (HS364) infected with O157:H7 with HUS (panelB). Lanes: [1]86-24 (O157:H7); [2] 84-289 (O157:H7); [3] 933 (O157:H7);[4] NF4 (O157:H7); [5] 83-574 (026:H11); [6] 6549 (O26:H11). Sera (bothdiluted 1:5000 final) were reacted to identical blots with equalloadings of antigen.

[0038]FIG. 4: Western immunoblot using serum from a cow prior toexperimental infection with E. coli 0157:H7 strains EDL933 and 86-24.Secreted Esp proteins from EDL933 and 86-24 strains were used asantigens and 25 weeks post-infection.

[0039]FIG. 5: Bovine antibody responses to EspB (2 ug/ml) purified fromE. coli O157 strain 93-111. Sera from two cattle (835 and M38) weretested on day 0 (uninfected) and 38 (835) or 25 (M38) weeks afterinfection.

[0040]FIG. 6: Immunoblot of culture supernatants from 84-289, 93-111,and 96-01 probed with human antiserum from an uninfected child (021),and HUS positive patient from the 1993 Washington state outbreak(HS364), and a patient from an isolated case of HUS (274-0013). Theantisera were diluted 1:10,000.

[0041]FIG. 7: Human antibody responses to EspB (2 ug/ml) purified fromO157:H7 E. coli. strain 93-111. HS366 and 274-0013 are from HUS patientsinfected with O157:H7 E. coli. 024 is a negative control.

DETAILED DESCRIPTION OF THE INVENTION

[0042] Enterohemorrhagic E. coli are the most common cause ofhemorrhagic colitis, a bloody diarrhea which can lead to lifethreatening hemolytic uremic syndrome (HUS) (1, 2). Infections caused byEHEC and another diarrheagenic pathogen, enteropathogenic E. coli (EPEC)result in a histopathology called attaching and effacing (AE) (5, 22,48).

[0043] Another feature common to both EPEC and EHEC is the presence of alarge chromosomal locus called the LEE (Locus for Enterocyte Effacement)which encodes all of the known virulence factors necessary for AElesions (7). One gene within the LEE, eaeA (E. coli attaching andeffacing), encodes an outer membrane protein called intimin which isrequired but not sufficient for AE lesion formation (5, 48, 49). Twoother genes within the EPEC LEE, espA, encoding a 25 kilodaltonpolypeptide (50) and espB (formerly called eaeB) encoding a 37kilodalton polypeptide (51) are necessary for epithelial cell signaltransduction events during AE lesion formation (52). The espA and espBgene products are secreted to the culture supernatant during in vitrogrowth and are presumed to interact with host cells during naturalinfections (53,54). When the espB gene is present, the eae genes arealso present (7). A transport apparatus that secretes the Esp proteinsand several other polypeptides is also encoded by genes in the EPEC andEHEC LEE (53).

[0044] The strain of EHEC used in the invention is not specificallyimportant. The E. coli 0157:H7 was chosen for the majority of studiessince this is the most common serotype implicated in disease in NorthAmerica. However results obtained with 0157:H7 can be related to otherserotypes of Stx-producing E. coli strains since non-0157:H7 serotypesare more prominent outside of North America. Strains used in this studyare included in Table 1.

[0045] Enterohemorrhagic E. coli strains secrete proteins in addition toStx antigens, but such antigens are previously undescribed. Theseproteins are produced in vivo during infection. Several of theseproteins are secreted from the bacterial cells by a specialized proteinsecretion system homologous to the secretion systems involved insecretion of crucial virulence factors by Shigella, Salmonella, andYersina species. These proteins have been purified sufficiently to beemployed in serological assays. This data demonstrates that a stongimmunological response is produced in humans and cows. The antibodiesare detected via Western blot and ELISA.

[0046] To obtain large quantities of highly purified protein, the genesencoding these highly antigenic proteins were sequenced. The choice ofmethod used to clone the genes which encode the antigenic proteins isnot crucial. Well-known methods in the art could be employed. Forexample, the preferred method of the invention is the Polymerase ChainReaction (PCR). For example, the primer sequences used in the PCRreaction for the espB gene are as follows: K-638,5′-CCATTTGCTCTTCCGCACCCAGCTAAGCGACC; (SEQ ID NO. 1) K-639,5′-TCCGACGCATATGAATACTATTGATAATACT. (SEQ ID NO. 2)

[0047] PCR primers were designed using homologous gene sequences whichencode secreted proteins from EPEC (51, 52). Restriction sites for Nde Iand Sap I were incorporated into the 5′ and 3′ ends of espBrespectively. The gene for the 37 kilodalton protein from EHEC wascloned using this method. Subsequent cloning and expression wasaccomplished using standard methods in the art.

[0048] The purification method used to isolate the recombinant proteinsis not crucial to the invention. Any known method can be used to isolateand sufficiently purify the recombinant protein of interest. Suchmethods are known in the art. For example, the preferred embodiment isthe IMPACT system (New England Biolabs). This system allows therecombinant protein to be readily purified by column chromatography,however, one of ordinary skill in the art would know appropriate proteinpurification techniques which can also be used.

[0049] Using these standard procedures, the gene encoding the 37kilodalton protein, also known as EspB was cloned and sequenced. Thegene sequence which encodes the EspB protein as shown is as follows:           10                  30                  50ATGAATACTATTGATAATACTCAAGTAACGATGGTTAATTCCGCTTCGGAGAGTACGACC (SEQ ID #4)M  N  T  I  D  N  T  Q  V  T  M  V  N  S  A  S  E  S  T  T           70                  90                 110GGCGCTTCCAGTGCAGTTGCCGCATCTGCTTTATCAATTGATTCATCTCTGCTTACTGATG  A  S  S  A  V  A  A  S  A  L  S  I  D  S  S  L  L  T  D          130                 150                 170GGTAAGGTTGATATTTGTAAGCTGATGCTGGAAATTCAAAAACTCCTCGGCAAGATGGTGG  K  V  D  I  C  K  L  M  L  E  I  Q  K  L  L  G  K  M  V          190                 210                 230ACTCTATTGCAGGATTACCAACAAAAACAATTGGCGCAAAGCTATCAGATTCAGCAGGCCT  L  L  Q  D  Y  Q  Q  K  Q  L  A  Q  S  Y  Q  I  Q  Q  A          250                 270                 290GTTTTTGAGAGCCAGAATAAAGCTATTGAGGAAAAAAAAGCCGCGGCAACCGCTGCTTTGV  F  E  S  Q  N  K  A  I  E  E  K  K  A  A  A  T  A  A  L          310                 330                 350GTTGGCGGGATTATTTCATCAGCATTGGGGATCTTAGGTTCTTTTGCAGCAATGAACAACV  G  G  I  I  S  S  A  L  G  I  L  G  S  F  A  A  M  N  N          370                 390                 410GCGGCTAAAGGGGCTGGTGAGATTGCTGAAAAAGCAAGCTCTGCATCTTCAAAGGCTGCTA  A  K  G  A  G  E  I  A  E  K  A  S  S  A  S  S  K  A  A          430                 450                 470GGTGCGGCTTCTGAGGTTGCAAATAAAGCTCTGGTCAAGGCTACGGAAAGTGTTGCTGATG  A  A  S  E  V  A  N  K  A  L  V  K  A  T  E  S  V  A  D          490                 510                 530GTCGCAGAGGAGGCATCCAGTGCGATGCAGAAAGCGATGGCCACAACAACGAAAGCAGCCV  A  E  E  A  S  S  A  M  Q  K  A  M  A  T  T  T  K  A  A          550                 570                 590AGCCGTGCATCTGGCGTTGCAGATGATGTTGCGAAAGCCTCTGACTTTGCTGAAAATCTTS  R  A  S  G  V  A  D  D  V  A  K  A  S  D  F  A  E  N  L          610                 630                 650GCAGACCCCGCCGAGAAGACAAGCAGAATCAATAAGTTGTTGAATTCCGTACATAAACTGA  D  P  A  E  K  T  S  R  I  N  K  L  L  N  S  V  D  K  L          670                 690                 710ACCAATACCACAGCATTTGTTGCCGTGACCAGTCTTGCTGAAGGTACGAAAACGTTGCCAT  N  T  T  A  F  V  A  V  T  S  L  A  E  G  T  K  T  L  P          730                 750                 770ACAACAATATCTGAGTCCGTCAAATCGACTCATGAGGTTAATGAACAACGTGCGAAGTCGT  T  I  S  E  S  V  K  S  T  H  E  V  N  E  Q  R  A  K  S          790                 810                 830CTGGAAAACTTCCAGCAGGGGAATCTGGAGCTGTATAAACAAGACGTTCGCAGAACGCAGL  E  N  F  Q  Q  G  N  L  E  L  Y  K  Q  D  V  R  R  T  Q          850                 870                 890GATGATATCACGACTCGTCTGCGTGATATAACGTCCGCTGTCCGCGATCTCCTTGAGGTCD  D  I  T  T  R  L  R  D  I  T  S  A  V  R  D  L  L  E  V          910                 930 CAGAATCGTATGGGGCAATCGGGTCGCTTAGCTGGGTQ  N  R  M  G  Q  S  G  R  L  A  G

[0050] The antigenic proteins induce antibodies in HUS patients. The 24,37, and 110 kilodaltons are the most consistently seen, and representthe three seemingly most antigenic proteins in the strains studied. The37 kilodalton protein is also known as EspB; the 24 kilodalton is knownas EspA These proteins are the most preferred embodiments for use in theinvention.

[0051] Western immunoblots and enzyme-linked immunosorbent assays(ELISA) were used to identify the serodiagnostic capabilities of thenewly discovered secreted antigens from EHEC. Briefly, Western blotswere done as follows. The antigen (either 37 or 24 kilodalton) sample isseparated in an analytical gel, for example an SDS polyacrylamide gel oran isoelectric focusing gel. The resolved molecules are transferredelectrophoretically to a nitrocellulose membrane in a blotting tank. Theblot is then treated sequentially with serum collected from a subject,and washed, and then a radiolabelled or chemiluminescent conjugate todetect antibodies is bound to the blot. After washing again, the blot isplaced in contact with X-ray flm in a cassette; the autoradiograph isdeveloped and the antigen bands which have bound the antibody arevisible.

[0052] ELISA assays were performed as follows. The antigen in saline isincubated on a plastic plate, and small quantities become absorbed ontothe plastic surface. Free antigen is washed away. The plate may then beblocked with excess of an irrelevant protein to prevent any subsequentnon-specific binding of proteins. Test antibody is added, which binds tothe antigen. Unbound proteins are washed away and a ligand is addedwhich can detect the antibody and is covalently coupled to an enzymesuch as peroxidase. The bound ligand is visualized by the addition of anenzymatic substrate, which acts as a colored indicator.

[0053] Sera from cows experimentally infected with E. coli 0157:H7 wereexamined (55). As is extensively documented in the literature, cows arethe major reservoir of this pathogenic bacteria and people become illfrom eating insufficiently cooked hamburgers or vegetables which havecome in contact with cow feces. However, there is no reliable way todetermine which cows have this pathogen in their intestinal tract.Simple bacteriologic examination of feces is very insensitive. Theinvention demonstrates that when cows harbor this organism in theirintestine they develop antibodies to the secreted proteins. Westernimmunoblot using serum from a cow that was collected before experimentalinfection with E. coli 0157:H7 (day 0 serum) and serum collected fromthe same cow 25 or 38 weeks after infection. The immunoblot shows thatthere are no antibodies at day 0, but there is a strong antibody resonseto these proteins 25 or 38 weeks after infection.

[0054] A test kit is envisioned to test beef cows in a herd. Cows whichare positive would not then be used to make hamburger or other foods forhuman consumption. The purified 37 kilodalton protein could be used in atest where the presence of antibodies could turn an indicator a certaincolor. Any kind of substrate could be used. The substrate, such asfilter paper would contian secreted Esp protein. Serum would be added tothe substrate and a color indicator. A color indicator could be used todetect the presence of antibodies in a sample of serum from a subject.Different kits would be developed for cow and human sera.

[0055] The following examples are provided for illustrative purposesonly, and are in no way intended to limit the scope of the presentinvention.

EXAMPLE 1 Secretion of Proteins of E. coli 0157:H7

[0056]E. coli 0157:H7 were grown in 100 ml of Eagles's minimal medium at37° C. shaking to mid-log (OD₆₀ of 1.0). Bacteria were pelleted bycentrifugation (10,000×g, 10 min.) and phenylmethylsulfonyl fluoride(PMSF, 50 ug/ml), aprotinin (0.5 ug/ml) and EDTA (0.5 uM) were added tothe supernatants to inhibit digestion by proteases. After passagethrough 0.45 um filters, the culture supernatants were concentrated 100fold in Omegacell disposable stirred cell filtration devices (FiltronTechnology, Northborough, Mass.). Samples were then run on SDS-PAGE gelsand stained (Millipore) and processed for immunoblots as previouslydescribed (53).

[0057] Samples were analyzed by SDS-PAGE or western immunoblot usingrabbit antiserum (diluted 1:1000) raised against EPEC secreted proteinsor human serum (diluted 1:1000) from an individual who developed HUSsubsequent to an 0157:H7 EHEC infection.

EXAMPLE 2 Detection of EHEC Secreted Proteins With EPEC Antiserum

[0058] Six EHEC strains were tested for the presence of secretedproteins using antiserum raised against EPEC secreted proteins. FIGS. 1Aand 1B shows an SDS PAGE and an identical western immunoblot of thesecreted proteins from five 0157:H7 stains (lanes 1-5) and two 026:H11strains (lanes 6-7). Culture supernatants from EPEC strain E2348/69(Lane 8) and E. coli HS-4 (lane 9), an avirulent fecal isolate, areshown for comparison. All of the EHEC secrete 100-110, 37- and24-kilodalton polypeptides with the exception of 6549 which does notsecrete the 100-110 kilodalton protein.

EXAMPLE 3 Detection of EHEC Secreted Proteins With HUS Human Serum

[0059] Westerm immunoblot analysis was performed to determine whetherthe secreted proteins are present in human serum collected from apatient who developed HUS as a result of an 0157:H7 EHEC infection. FIG.2 shows that the EHEC secreted proteins are recognized by human serumand that the recognition pattern is similar to that seen with rabbitantiserum against EPEC secreted proteins. All of the strains secretepolypeptides of 37- and 24-kilodaltons that are recognized by the humanserum from an HUS patient but not with serum from uninfected children.The results are presented in FIG. 6.

EXAMPLE 4 Immunoblot of Secreted EHEC Proteins Probed With Serum fromHUS+/−Patients

[0060] Sera was screened from 6 patients infected with E. coli 0157:H7during the 1993 Washinton State outbreak. Of these 6 sera, 3 were fromchildren with HUS and 3 were from children infected with 0157:H7 and haddiarrhea but no HUS. With some serum samples, we can dilute the sera togreater than 1:100,000 and still see a clear response to these secretedEHEC proteins. An example of this is shown in FIG. 3 in which secretedproteins from the identical panel of strains were immunoblotted withserum from an HUS-negative child (panel A) and from an HUS-positivechild (panel B).

EXAMPLE 5 Cloning of espB Gene from E. coli 0157:H7

[0061] The espB gene of E. coli 0157:H7 strain 93-111 (encoding 37kilodalton protein) was generated using the polymerase chain reactionwith restriction sites that allowed the gene to be cloned into a vectorpCYB1 from the IMPACT I, One Step protein Purification System (NewEngland Biolabs). The primer sequences used for PCR were as follows:K-638, 5′-CCATTTGCTCTTCCGCACCCAGCTAAGCGACC; (SEQ ID No. 1) K-639,5′-TCCGACGCATATGAATACTATTGATAATACT. (SEQ ID No. 2)

[0062] PCR primers were designed using homologous gene sequences whichencode secreted proteins from EPEC. Restriction sites for Nde I and SapI were incorporated into the 5′ and 3′ ends of espB respectively. Thesequence of the PCR product was confirmed using the above primers tosequence the DNA before cloning. After the gene was cloned into pCYB1 itwas sequenced again using the universal vector primers NEB #1260 and NEB#1261 supplied with the IMPACT kit.

EXAMPLE 6 Purification of the espB Protein

[0063] The IMPACT I Intein Mediated Purification with an AffinityChitin-binding Tag is a protein purification system that allows thecloning of genes into vectors supplied with the kit to generate a threepart gene fusion (C-terminal) which can be over expressed by inducing atac promotor with IP>TG (0.5 mM) and then purified. The gene ofinterest, the gene encoding the 37 kilodalton (espB) is cloned into theappropriate vector so that it is in frame with an Intein moiety and achitin binding domain (CBD). The vectors are under the control of theptac promotor which enables IPTG induced expression of the fusionprotein. The induced bacterial culture is then lysed by a French Pressand the extract containing EspB-intein-CBD-fusion is added to a columncontaining chitin beads. The EspB protein is then cleaved from thefusion with dithiothreitol (30 mM) and eluted from the column. Thedithiothreitol was removed by dialysis against phosphate buffered saline(4 times 4 liters) and concentrated with Centricon concentrators(Amicon).

[0064] N-terminal sequencing of the EspB protein (37 kilodalton) wasdetermined by the Edman degradation procedure. The first four aminoacids of the 37 kilodalton from the EHEC strain 84-289 supernatants areMet-Asn-Thr-Ile (SEQ ID No. 3).

EXAMPLE 7 Western Immunoblot Using Infected Cow Sera

[0065] Western immunoblots prepared according to the procedure outlinedin Example 1 were used with serum from a cow that was collected beforeexperimental infection with E. coli 0157:H7 (day 0 serum) and serum fromthe same cow collected 25 weeks after infection (55). The results arepresented in FIG. 4. The two different gel lanes labeled EDL933 and86-24 are antigens prepared from two different strains of E. coli0157:H7, EDL933 and 86-24.

EXAMPLE 8 Enzyme-Linked Immunosorbent Assay (ELISA)

[0066] Sera was obtained from two cows that had been experimentallyinfected with E. coli 0157:H7. From one cow, sera from week 0 (beforeinfection) and week 38 (after infection are shown. From the other cow,sera from week 0 and week 25.

[0067] Immulon-II (Dynatech) plates were coated overnight with 100 ul ofpurified EspB (2 ug/ml) in phosphate buffered saline (PBS). Controlwells contain an equal volume of PBS. The plates were washed three timeswith PBS and unbound sites were blocked with 6% bovine serum albumin,0.05% Tween by incubation for one hour at room temperature. Plates wereincubated for 1 hour at 37° C. with dilutions of the following sera:Bovine M38 Day 0, M38 25 weeks after infection, 835 Day 0, 835 38 weekspost-infection. Bovine anti-EspB responses were detected with a goatanti-bovine horseradish peroxidase conjugate (1:5000). The conjugateincubations were for 1 hour at 37° C. in PBS/Tween. The secondaryantibodies were removed by washing and antibody responses detected withthe appropriate substrate solution for 30 minutes and read at theappropriate wavelength on an ELISA plate reader. Data represent the meanof duplicate antibody dilutions after subtracting the values of thenegative control wells.

[0068]FIG. 5 shows different dilutions of the cow sera on the bottomaxis. The white bar is the absorbance from week 0 sera and the black baris the absorbance at week 25 or 38. Even diluting the cow sera to 1:200,there is still a clear-cut difference between the pre- andpost-infection titers.

EXAMPLE 9 Human Antibody Responses Using an ELISA Test

[0069] Sera was obtained from two HUS patients infected with O157:H7 E.coli. EspB (2 ug/ml) were purified from E. coli O157:H7 strain 93-111.HS366 and 274-0013 are from HUS patients infected with O157:H7 E. coli.Procedures were followed as in example 8. Results are shown in FIG. 7.

1 5 1 33 DNA Artificial Sequence PCR Primers for cloning Escherichiacoli Secreted Protein B 1 ccatttgctc ttccgccacc cagctaagcg acc 33 2 31DNA Artificial Sequence PCR Primer for cloning Escherichia coli SecretedProtein B 2 tccgacgcat atgaatacta ttgataatac t 31 3 4 PRT Escherichiacoli 3 Met Asn Thr Ile 1 4 937 DNA Escherichia coli 4 atgaatactattgataatac tcaagtaacg atggttaatt ccgcttcgga gagtacgacc 60 ggcgcttccagtgcagttgc cgcatctgct ttatcaattg attcatctct gcttactgat 120 ggtaaggttgatatttgtaa gctgatgctg gaaattcaaa aactcctcgg caagatggtg 180 actctattgcaggattacca acaaaaacaa ttggcgcaaa gctatcagat tcagcaggcc 240 gtttttgagagccagaataa agctattgag gaaaaaaaag ccgcggcaac cgctgctttg 300 gttggcgggattatttcatc agcattgggg atcttaggtt cttttgcagc aatgaacaac 360 gcggctaaaggggctggtga gattgctgaa aaagcaagct ctgcatcttc aaaggctgct 420 ggtgcggcttctgaggttgc aaataaagct ctggtcaagg ctacggaaag tgttgctgat 480 gtcgcagaggaggcatccag tgcgatgcag aaagcgatgg ccacaacaac gaaagcagcc 540 agccgtgcatctggcgttgc agatgatgtt gcgaaagcct ctgactttgc tgaaaatctt 600 gcagaccccgccgagaagac aagcagaatc aataagttgt tgaattccgt agataaactg 660 accaataccacagcatttgt tgccgtgacc agtcttgctg aaggtacgaa aacgttgcca 720 acaacaatatctgagtccgt caaatcgact catgaggtta atgaacaacg tgcgaagtcg 780 ctggaaaacttccagcaggg gaatctggag ctgtataaac aagacgttcg cagaacgcag 840 gatgatatcacgactcgtct gcgtgatata acgtccgctg tccgcgatct ccttgaggtc 900 cagaatcgtatggggcaatc gggtcgctta gctgggt 937 5 312 PRT Escherichia coli 5 Met AsnThr Ile Asp Asn Thr Gln Val Thr Met Val Asn Ser Ala Ser 1 5 10 15 GluSer Thr Thr Gly Ala Ser Ser Ala Val Ala Ala Ser Ala Leu Ser 20 25 30 IleAsp Ser Ser Leu Leu Thr Asp Gly Lys Val Asp Ile Cys Lys Leu 35 40 45 MetLeu Glu Ile Gln Lys Leu Leu Gly Lys Met Val Thr Leu Leu Gln 50 55 60 AspTyr Gln Gln Lys Gln Leu Ala Gln Ser Tyr Gln Ile Gln Gln Ala 65 70 75 80Val Phe Glu Ser Gln Asn Lys Ala Ile Glu Glu Lys Lys Ala Ala Ala 85 90 95Thr Ala Ala Leu Val Gly Gly Ile Ile Ser Ser Ala Leu Gly Ile Leu 100 105110 Gly Ser Phe Ala Ala Met Asn Asn Ala Ala Lys Gly Ala Gly Glu Ile 115120 125 Ala Glu Lys Ala Ser Ser Ala Ser Ser Lys Ala Ala Gly Ala Ala Ser130 135 140 Glu Val Ala Asn Lys Ala Leu Val Lys Ala Thr Glu Ser Val AlaAsp 145 150 155 160 Val Ala Glu Glu Ala Ser Ser Ala Met Gln Lys Ala MetAla Thr Thr 165 170 175 Thr Lys Ala Ala Ser Arg Ala Ser Gly Val Ala AspAsp Val Ala Lys 180 185 190 Ala Ser Asp Phe Ala Glu Asn Leu Ala Asp ProAla Glu Lys Thr Ser 195 200 205 Arg Ile Asn Lys Leu Leu Asn Ser Val AspLys Leu Thr Asn Thr Thr 210 215 220 Ala Phe Val Ala Val Thr Ser Leu AlaGlu Gly Thr Lys Thr Leu Pro 225 230 235 240 Thr Thr Ile Ser Glu Ser ValLys Ser Thr His Glu Val Asn Glu Gln 245 250 255 Arg Ala Lys Ser Leu GluAsn Phe Gln Gln Gly Asn Leu Glu Leu Tyr 260 265 270 Lys Gln Asp Val ArgArg Thr Gln Asp Asp Ile Thr Thr Arg Leu Arg 275 280 285 Asp Ile Thr SerAla Val Arg Asp Leu Leu Glu Val Gln Asn Arg Met 290 295 300 Gly Gln SerGly Arg Leu Ala Gly 305 310

We claim:
 1. A purified protein called EspB isolated from EHEC having a molecular weight of 37 kilodaltons.
 2. The protein of claim 1 which is isolated from E. coli 0157:H7.
 3. The protein of claim 2 which has an N-terminal amino acid sequence of Met-Asn-Thr-Ile (SEQ ID NO. 3).
 4. A purified protein called EspA isolated from E. coli having a molecular weight of 24 kilodaltons.
 5. The protein of claim 4 which is isolated from E. coli 0157:H7.
 6. A DNA molecule encoding the protein of claim
 1. 7. The DNA molecule of claim 6 having the DNA sequence          10                  30                  50 ATGAATACTATTGATAATACTCAAGTAACGATGGTTAATTCCGCTTCGGAGAGTACGACC (SEQ ID No. 4) M  N  T  I  D  N  T  Q  V  T  M  V  N  S  A  S  E  S  T  T                  70                  90                 110 GGCGCTTCCAGTGCAGTTGCCGCATCTGCTTTATCAATTGATTCATCTCTGCTTACTGAT                 130                 150                 170 GGTAAGGTTGATATTTGTAAGCTGATGCTGGAAATTCAAAAACTCCTCGGCAAGATGGTG G  K  V  D  I  C  K  L  M  L  E  I  Q  K  L  L  G  K  M  V                 190                 210                 230 ACTCTATTGCAGGATTACCAACAAAAACAATTGGCGCAAAGCTATCAGATTCAGCAGGCC T  L  L  Q  D  Y  Q  Q  K  Q  L  A  Q  S  Y  Q  I  Q  Q  A                 250                 270                 290 GTTTTTGAGAGCCAGAATAAAGCTATTGAGGAAAAAAAAGCCGCGGCAACCGCTGCTTTG V  F  E  S  Q  N  K  A  I  E  E  K  K  A  A  A  T  A  A  L                 310                 330                 350 GTTGGCGGGATTATTTCATCAGCATTGGGGATCTTAGGTTCTTTTGCAGCAATGAACAAC V  G  G  I  I  S  S  A  L  G  I  L  G  S  F  A  A  M  N  N                 370                 390                 410 GCGGCTAAAGGGGCTGGTGAGATTGCTGAAAAAGCAAGCTCTGCATCTTCAAAGGCTGCT A  A  K  G  A  G  E  I  A  E  K  A  S  S  A  S  S  K  A  A                 430                 450                 470 GGTGCGGCTTCTGAGGTTGCAAATAAAGCTCTGGTCAAGGCTACGGAAAGTGTTGCTGAT G  A  A  S  E  V  A  N  K  A  L  V  K  A  T  E  S  V  A  D                 490                 510                 530 GTCGCAGAGGAGGCATCCAGTGCGATGCAGAAAGCGATGGCCACAACAACGAAAGCAGCC V  A  E  E  A  S  S  A  M  Q  K  A  M  A  T  T  T  K  A  A                 550                 570                 590 AGCCGTGCATCTGGCGTTGCAGATGATGTTGCGAAAGCCTCTGACTTTGCTGAAAATCTT S  R  A  S  G  V  A  D  D  V  A  K  A  S  D  F  A  E  N  L                 610                 630                 650 GCAGACCCCGCCGAGAAGACAAGCAGAATCAATAAGTTGTTGAATTCCGTAGATAAACTG A  D  P  A  E  K  T  S  R  I  N  K  L  L  N  S  V  D  K  L                 670                 690                 710 ACCAATACCACAGCATTTGTTGCCGTGACCAGTCTTGCTGAAGGTACGAAAACGTTGCCA T  N  T  T  A  F  V  A  V  T  S  L  A  E  G  T  K  T  L  P                 730                 750                 770 ACAACAATATCTGAGTCCGTCAAATCGACTCATGAGGTTAATGAACAACGTGCGAAGTCG T  T  I  S  E  S  V  K  S  T  H  E  V  N  E  Q  R  A  K  S                 790                 810                 830 CTGGAAAACTTCCAGCAGGGGAATCTGGAGCTGTATAAACAAGACGTTCGCAGAACGCAG L  E  N  F  Q  Q  G  N  L  E  L  Y  K  Q  D  V  R  R  T  Q                 850                 870                 890 GATGATATCACGACTCGTCTGCGTGATATAACGTCCGCTGTCCGCGATCTCCTTGAGGTC D  D  I  T  T  R  L  R  D  I  T  S  A  V  R  D  L  L  E  V                 910                 930 CAGAATCGTATGGGGCAATCGGGTCGCTTAGCTGGGT. Q  N  R  M  G  Q  S  G  R  L  A  G


8. A DNA molecule encoding the protein of claim
 4. 9. A method for diagnosing whether a subject has been infected with EHEC comprising the steps: (1) obtaining a body fluid or tissue sample from a subject who is suspected of being infected with EHEC, (2) using the Esp protein, immunologically determining the presence of antibodies which are specific to said Esp in the body fluid or tissue sample of step (1), (3) the presence of antibodies which are specific to said Esp indicating active infection with EHEC.
 10. The method of claim 9, wherein the Esp protein is 37 kilodalton.
 11. The method of claim 9, wherein the Esp protein is 24 kilodaltons
 12. The method of claim 9, wherein the EHEC is E. coli O157:H7.
 13. The method of claim 9, wherein the EHEC is Non-O157:H7 STEC.
 14. The method as claimed in claim 9, wherein said body fluid is selected from the group consisting of plasma, cerebrospinal fluid, saliva, semen, sweat, urine, and amniotic fluid.
 15. The method as claimed in claim 14, wherein said body fluid is plasma.
 16. The method as claimed in claim 9, wherein said tissue sample is selected from the group consisting of adrenal tissue, red blood cells, lymphocytes, and platelets.
 17. The method as claimed in claim 16, wherein said tissue sample is red blood cells.
 18. The method of claim 9, wherein said immunological determination is by a Western immunoblot.
 19. The method of claim 9, wherein said immunological determination is by an ELISA test.
 20. The method of claim 9, wherein said subject is a human.
 21. The method of claim 9, wherein said subject is a cow.
 22. A kit for diagnosing whether a subject has been infected with EHEC comprising (1) a container for holding a body fluid or tissue sample from said subject, (2) a substrate containing a purified Esp protein and an indicator which changes color in the presence of antibodies to said purified Esp protein.
 23. The kit of claim 22 wherein the Esp protein is 37 kilodaltons.
 24. The kit of claim 22 wherein the Esp protein is 24 kilodaltons.
 25. The method of diagnosing whether a subject has been infected with EHEC comprising using the kit of claim
 22. 26. The kit of claim 22, wherein said subject is a human.
 27. The kit of claim 22, wherein said subject is a cow.
 28. The kit of claim 22, wherein said substrate is a solid-phase matrix. 